RU2216350C2 - Strain of bacterium salmonella typhimurium = 14 used for preparing vaccines and diagnostic preparations - Google Patents

Abstract

FIELD: veterinary microbiology, vaccines. SUBSTANCE: strain of bacterium Salmonella typhimurium = 14 shows morphological, cultural and enzymatic properties that are typical for salmonella genus. The proposed strain by its serological properties is agglutinated with salmonellous monoreceptor sera 04 (1 and 2 complex) H-1. Strain is a producer of endotoxin and hemolysine. Strain shows protein structure, its nucleotide DNA composition (% GC) is 53.1, it comprises plasmids of molecular mass 64 mDa and it is lysed with salmonellous bacteriophage. Virulence LD₅₀ for white mice is 1.7 x 10⁶ live bacterial cells in intraperitoneal administration. Strain is pathogenic for calves and profuse diarrhea is noticed on 2-d day and animal death on 3-4-th day. Concentration of bacterial cells in the strain culturing is 40-50 billion cells by optical turbidity standard GISK and protective effect after infection with virulent strains occurs on 5-7-th day. The strain is useful for preparing vaccines against salmonellosis in animals and for preparing diagnostic preparation and preparing hyperimmunic curative sera and globulins also. EFFECT: valuable medicinal properties of strain and vaccine.

Description

 The invention relates to the field of veterinary microbiology, in particular to the production of a strain of Salmonella typhimurium suitable for use in vaccines and diagnostic preparations against salmonellosis of farm animals.

Cattle salmonellosis is an infectious disease characterized by bacteremia, toxemia, abortion, or the birth of unviable young animals. Young animals aged 10-30 days are most susceptible. In the acute course of the young, depression is manifested, refusal of food, fever up to 41 ^o C, then paresis of the forelimbs and with untimely treatment after 36-48 hours death occurs. With a prolonged course of the disease for 2-3 days, calves develop diarrhea.

 The disease spreads according to the type of enzootia, grows gradually and for a long time is kept at a high level. Often, up to 80-90% of calves are ill with a mortality rate of 20-80%.

 Damage consists of the cost of treatment, the death of young animals.

 The disease is registered in all regions of the country, in connection with this, the relevance of the fight against this disease becomes apparent.

Known strain of Salmonella typhimurium VGNKI 3 for the manufacture of "Vaccines against salmonellosis of young animals". The accumulation of microbial cells (MK) strain in the vaccine amounted to 30-45 billion in 1 cm ³ [1].

 Also known is a strain of Salmonella typhimurium 159, which is used for the manufacture of "Inactivated emulsion vaccine against calf salmonellosis."

The strain Salmonella typhimurium 159 has the following properties:
Cultural and morphological characters. Gram-negative, motile, small, slightly curved rods, forming on the MPA convex colonies 1-3 mm in diameter, transparent or whitish. They grow well on ordinary nutrient media, on the BCH they form a film and a parietal ring.

Physico-biological properties. Aerob, capable of growing in microaerobic conditions. The optimum pH of 7.2 at a temperature of 37 ^o C.

 Enzymatic properties. Does not change milk, does not thin gelatin, does not secrete indole, does not decompose urea; emits hydrogen sulfide, ferments glucose, mannitol, sorbitol, arabinose, inositol, maltose with the formation of acid; lactose and sucrose does not ferment; has enzymes lysine decarboxylase and ornithine decarbase.

 Agglutinating properties. It is agglutinated by salmonella serums: "O" - 1, 4, 5, 12; H-I; 12.

Pathogenic properties. Pathogenic for white mice: LD ₅₀ is 5x10 ³ cells with intraperitoneal infection [2].

 The objective of our research was to obtain a strain of Salmonella typhimurium, which has a stable low residual virulence, high immunogenicity and is suitable for use in the preparation of vaccines against salmonellosis of calves and fur animals, as well as for diagnostic preparations.

 The proposed strain of Salmonella typhimurium 14, the causative agent of salmonellosis, is assigned to the Enterobacteriaceae family, to the genus Salmonella, and was isolated from the piglet’s organs in the LLL in infectious pathology and biotechnology in 1988 in the Efremovsky pig farm in the Tula Region.

 The proposed strain was deposited on April 27, 2001, the registration number is “Salmonella typhimurium 14 strain - MGAVMiB - DEP” in the All-Russian State Collection of Microorganism Strains used in veterinary medicine and animal husbandry (123022, Russia, Moscow, 5 Zvenigorodskoye Shosse, VGNKI).

 The storage location of the production strain of Salmonella typhimurium 14, the causative agent of salmonellosis of calves, has been determined by the collection of the research laboratory of infectious pathology and biotechnology of Moscow State Medical Academy of Medicine and Biology. K.I. Skryabin (109472, Moscow, Ak. Skryabin St., 23), duplicate storage of the Research Institute of Microbiology of the Ministry of Defense of the Russian Federation (610024, Russia, Kirov, Oktyabrsky pr., 121, military unit 23527).

The proposed production strain of Salmonella typhimurium 14 has the following properties:
Morphological, cultural and enzymatic properties. Gram-negative motile polymorphic bacilli form translucent S-form colonies after 18-24 hours of growth on a solid nutrient medium at a temperature of 36-38 ^o C.

 Enzymatic activity. It ferments with the formation of acid and gas glucose, rhamnose, sorbitol, mannitol, does not ferment lactose, sucrose, maltose, inositol, does not hydrolyze urea, forms hydrogen sulfide, does not form indole and acetylmethylcarbinol in the Voges-Proskauer reaction.

 Serological properties. Agglutinates with salmonella serums 04 (1 and 2 complex) and H-I.

 Toxigenicity. The strain is a producer of endotoxins and hemolysins.

Virulent properties. Virulence of LD ₅₀ for white mice intraperitoneally <1.7x10 ⁶ live m.k.

 Pathogenic to calves. With oral infection of calves 10 billion m. 2-3 days noted profuse diarrhea, and the death of animals occurred on 2-4 days, with pathological anatomical changes characteristic of salmonellosis.

 Protein structure.

 The nucleotide composition of DNA (% HC) is 53.1.

 Contains plasmids with m.v. 64 MD.

 Lysed with salmonella polyvalent bacteriophage.

The applied method of storing and maintaining the strain. In a lyophilized state under vacuum in hermetically sealed ampoules at minus 18-20 ^o C after passage through the body of white mice.

 The frequency of reseeding on nutrient media 15-20 days.

 Transportation conditions. In a lyophilized state under vacuum in hermetically sealed ampoules.

 Shelf life 10 years.

Example. The strain Salmonella typhimurium 14 is grown in reactors in casein broth. The sowing dose is 150-300 million m.k. per 1 cm ^{3 of} nutrient medium.

The cultivation is carried out for 16-18 hours at a temperature of 36-37 ^o With constant stirring and intensive aeration, based on the volume of sterile air per volume of medium per minute (in a ratio of 1: 3).

 The concentration of microbial cells after 16-18 hours is 40-50 billion m. according to the optical turbidity standard GISK im. K.A. Tarasovich, which made it possible to apply the strain for the manufacture of a vaccine against salmonellosis of farm animals and fur animals.

 A vaccine made using the proposed strain provides a 90% protective effect against infection with virulent epizootic strains on days 5-7.

 The vaccine from the production strain Salmonella typhimurium 14 was tested with a positive result from 1995 to 2001. in Russia and the CIS countries: Belarus, Kyrgyzstan, Uzbekistan, etc. on 11550 heads of pregnant cows, 7290 pregnant sows, 4270 ewes, 2290 fur animals.

 The safety of the livestock is 96-99%.

 The proposed strain can be used for the production of salmonella vaccines and diagnosticums, as well as for the production of hyperimmune therapeutic serums and globulins.

Sources of information:
1. RF patent 1197187, cl. A 61 K 39/112, 1998

 2. RF patent 2154495, cl. A 61 K 39/112, C 12 N 1/20, 2000

Claims (1)

 The bacterial strain Salmonella typhimurium VGNKI 14 - MGAVM and B-DEPT, used for the manufacture of vaccines and diagnostic drugs.